Nanocrystals are fluorescent particles that are widely used to study biochemical and even biological systems, because they are easily visualized and tracked. They serve as labels that are easily observed by their fluorescence emissions, and permit a user to track them to study the location, transport, or environment of biochemicals or cells that are attached to a nanocrystal. Or a nanocrystal can be linked to a specific affinity agent like an antibody, and can then be used to visualize the corresponding antigen, to learn about its location, transport or environment. Because of their widespread use in biochemical and biological systems, it is important to make nanocrystals compatible with those systems. One aspect of compatibility is water solubility: while a nanocrystal is a particle that does not truly dissolve, it behaves in many ways like a soluble molecule because of its small size. Thus a nanoparticle that has a surface adapted to be compatible with water often behaves as though it were soluble in water, and will at times be referred to herein as water-soluble even though it may more properly be viewed as water-dispersable.
Methods for making core/shell nanocrystals that are fluorescent and have hydrophobic surfaces are well known. The hydrophobic surfaces of these nanocrystals typically result from a coating of hydrophobic passivating ligands such as trioctylphosphine (TOP), trioctylphosphine oxide (TOPO), oleic acid, octylphosphonic acid (OPA) or tetradecylphosphonic acid (TDPA) on the surface of the particle. These hydrophobic passivating ligands serve many roles, including, but not limited to: protecting the surface of the nanocrystal by keeping reactive molecules (and even the solvent) away from the nanocrystal surface, prevent the coalescence of multiple nanoparticles, prevent “dangling bonds and other similar surface defects that could serve as trap sites for an excited electron or hole and thereby promote non-radiative recombination (i.e., reduce the quantum yield), or protect the nanocrystal from reactions that could occur when it is in a photoactivated state. These ligands provide a layer of alkyl groups that form the solvent-exposed surface surrounding the nanocrystal, and thus they render the nanocrystal effectively hydrophobic, regardless of the properties of the nanocrystal surface itself. These intimately associated ligands and the nanocrystal they are associated with form a nanoparticle. Because the surface of the nanocrystal has many binding sites for such ligands, these ligands are packed onto the surface of the nanocrystal to form a surface layer of ligand molecules. This typically results in coating most or all of the exposed surface of the nanocrystal with a layer of alkyl groups hanging off of the ligands, and produces a nanocrystal with a surface that is very hydrophobic, i.e., incompatible with water.
Several ways of modifying nanocrystals to make them more water-soluble have been reported. One successful approach involves using the hydrophobic nature of the exposed surface to adhere a hydrophilic moiety. Adams, et al. (U.S. Pat. No. 6,649,138) used this approach: they constructed amphiphilic polymers having polar groups (carboxylates) and long-chain alkyl groups (hydrophobic domains), and introduced these amphiphilic polymers onto the hydrophobic surface of conventional nanocrystals. The hydrophobic domains of the AMPs ‘stick’ to the hydrophobic surface layer of the nanoparticles, exposing the polar carboxylates of the AMP to the exterior environment. This makes nanoparticles that have an outermost surface that is sufficiently polar to make the nanoparticle water soluble. This approach works well for certain applications, but it results in adding an additional layer on the outside of a nanocrystal, so it actually makes the nanoparticle larger.
Others have approached this problem by replacing the typical phosphine/phosphine oxide or other hydrophobic ligands with smaller moieties that do not have the long, hydrophobic alkyl groups that produce a hydrophobic surface on a conventional nanocrystal. Naasani, et al., for example, produced nanocrystals having relatively polar dipeptides on their surfaces. U.S. Pat. No. 6,955,855. These dipeptides use a binding group, e.g., imidazole ring, to coordinate to the nanocrystal surface, and they have a carboxylate and an amine in addition to the binding group that can be free to promote water solubility and/or to participate in crosslinking. This provided a much smaller nanoparticle than those of Adams, et al., and also provided a surface that was sufficiently polar to make the nanoparticle water-dispersable.
For some applications, there are certain advantages to making a nanoparticle as small as possible, especially for certain biological applications. For example, smaller particles diffuse more rapidly, have less effect on a molecule they are attached to, and may have less tendency to accumulate in specific tissues in vivo, where larger particles seem to get trapped by ‘filtration’ effects. See, e.g., Ballou, et al., Bioconjugate Chem., vol. 15, 79-86 (2004). Thus better methods for making nanocrystals into water-soluble nanoparticles are needed, preferably methods that keep these nanoparticles as small as possible while making them highly stable and maintaining their essential fluorescence characteristics. This disclosure provides methods for achieving such objectives, and thus provides compositions and methods that produce improved nanoparticles, especially small, stable, water-soluble ones.